Method and kit for quantitatively measuring beta-glucuronidase in multiple samples by enzyme-linked immunosorbent assay (ELIASA) instrument

A technology of glucuronidase and glucuronic acid, which is applied in the field of biochemical analysis, can solve the problems of inability to detect, decrease in enzyme activity, and long time consumption, and achieve high accuracy of standard products, less reagent usage, and reduced operating steps Effect

Inactive Publication Date: 2014-09-10
北京洛奇医学检验实验室股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 1. A large amount of reagents are used
[0006] 2. A large number of samples are required
[0007] In addition, ultraviolet spectrophotometer and automatic biochemical analyzer require a large amount of samples. If the sample size is not enough, the detection cannot be carried out.
[0008] 3. It takes a long time
[0011] In addition to the inaccurate results caused by the long time-consuming of the automatic biochemical instrument and the UV spectrophotometer, there may also be the possibility of cross-contamination of samples or standards
[0012] 4. Cleaning steps required
[0014] 5. Inaccurate results
Since the absorbance of the standard curve (usually five points) is not read at the same time, the time for detecting the enzyme activity is not synchronized, and the time extension will reduce the detected enzyme activity, so the standard curve obtained will be different, and is an unavoidable systematic error
In addition, the sampling and testing of each sample is a manual operation, and there will be operational errors
[0016] The detection of samples by the automatic biochemical instrument is better than that of the ultraviolet spectrop...

Method used

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  • Method and kit for quantitatively measuring beta-glucuronidase in multiple samples by enzyme-linked immunosorbent assay (ELIASA) instrument
  • Method and kit for quantitatively measuring beta-glucuronidase in multiple samples by enzyme-linked immunosorbent assay (ELIASA) instrument
  • Method and kit for quantitatively measuring beta-glucuronidase in multiple samples by enzyme-linked immunosorbent assay (ELIASA) instrument

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] The relationship between the concentration of embodiment 1β-glucuronidase and the absorbance of phenolphthalein glucuronic acid compound

[0090] 1. Purpose of the experiment:

[0091] To explore whether there is a linear relationship between the concentration of β-glucuronidase in feces and its relationship with phenolphthalein glucuronides.

[0092] 2. Materials and methods:

[0093] 1. β-glucuronidase detection principle: By extracting β-glucuronidase from stool samples, and reacting it with phenolphthalein glucuronic acid, and terminating the reaction with alkaline solution, a chromogenic complex is obtained.

[0094] 2. Glycine: purchased from sigma company. Use DDW to prepare according to 0.75g: 25mL, and store in separate packages for later use.

[0095] 3. Configure β-glucuronidase to a storage concentration of 10000U / mL, and then dilute it to 5000U / mL, 2500U / mL, 1250U / mL, 625U / mL, 312.5 / mL, 156.25U / mL, 78.125U / mL, 39.0625U / mL.

[0096] 4....

Embodiment 2

[0103] Embodiment 2 microplate reader method is to the determination of the linear range of β-glucuronidase quantification and limit of quantification and detection sensitivity

[0104] 1. Materials and methods

[0105] 1. Prepare the β-glucuronidase standard product to a storage concentration of 10000U / mL, and then dilute it with 75mM PBS to 5000U / mL, 2500U / mL, 1250U / mL, 625U / mL, 312.5U / mL, 156.25U / mL mL, 78.125U / mL, 39.0625U / mL and other concentrations for storage.

[0106] 2. Mix 0.01mL of the diluted β-glucuronidase standard solution with 0.09mL of the diluted phenolphthalein glucuronide solution, and transfer to a 96-well microplate. Other operations are the same as 5 and 6 in the implementation example 1.

[0107] 2. Experimental results:

[0108] 1. Determination of the linear range of β-glucuronidase by microplate reader

[0109] In order to determine the linear range of β-glucuronidase quantification by microplate reader method, β-glucuronidase w...

Embodiment 3

[0114] Embodiment 3 microplate reader method and ultraviolet method measure β-glucuronidase comparison

[0115] 1. Purpose of the experiment

[0116] 1. Simultaneously measure and compare with the microplate reader method of the present invention and the ultraviolet spectrophotometer to be measured β-glucuronidase sample, to prove the feasibility and accuracy of this patent method.

[0117] 2. Materials and methods

[0118] 2. The β-glucuronidase to be tested was purchased from Sigma Company.

[0119] 3. Use the standard curve method to quantify the β-glucuronidase in the side sample

[0120] (1) Standard product treatment: Dilute the β-glucuronidase standard product to 8 concentrations by the doubling dilution method, react with phenolphthalein glucuronic acid, add alkaline glycine to terminate the reaction, and read the absorbance value.

[0121] (2) Preparation of β-glucuronidase standard solution

[0122]Place the β-glucuronidase standard substance on an electronic bal...

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Abstract

The invention relates to a method and a kit for quantitatively measuring the beta-glucuronidase in multiple samples by an enzyme-linked immunosorbent assay (ELIASA) instrument. The kit internally comprises beta-glucuronidase standard substance, working solution, phenolphthalein glucuronic acid reaction liquid, termination reaction reagent, a pH meter and the like. The method and the kit have the advantages that the concentration of the beta-glucuronidase in the multiple samples can be detected at the same time, and shorter detection time is consumed; the sensitivity is high, and the detection limit is 39.0625U/ mL; the method is wide in linear range, the detection concentration is within the range of 39.0625-12500U/mL, and the three order of magnitudes are spanned; the accuracy is high; less reagent and fewer samples are used; the kit is convenient to operate; the samples can be fed once without being fed for a plurality of times; the cleaning step is omitted, so that the operation steps are reduced, cross contamination is prevented, and the result is guaranteed to be credible; the detected samples can be well stored so as to be repeatedly detected.

Description

Technical field: [0001] The invention belongs to the field of biochemical analysis, in particular to a method and a detection kit for quantitatively and simultaneously detecting β-glucuronidase in a plurality of samples with a microplate reader. Background technique: [0002] β-glucuronidase is a lysosomal enzyme that promotes the hydrolysis of the β-D-glycosidic bonds of many glucosides in the body. [0003] In the current technical method, the method for detecting β-glucuronidase in feces is limited to ultraviolet spectrophotometer and automatic biochemical analyzer. [0004] Both methods share the following disadvantages: [0005] 1. A large amount of reagents are used [0006] 2. Need more samples [0007] In addition, ultraviolet spectrophotometers and automatic biochemical analyzers require a large amount of samples. If the sample size is not enough, the detection cannot be performed. [0008] 3. It takes a long time [0009] When multiple samples need to be detec...

Claims

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Application Information

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IPC IPC(8): G01N33/573G01N21/31
CPCG01N33/573G01N33/54373G01N33/54393
Inventor 侯艳艳
Owner 北京洛奇医学检验实验室股份有限公司
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